Typically, cancer is treated with conventional treatment regimens such as surgery, hormonal therapies, chemotherapies, radiotherapies and/or other therapies. However, in many cases cancers which often are characterized by an advanced stage cannot be cured with present therapies. Despite progress in conventional cancer treatment regimens, metastatic disease essentially remains incurable and new treatment alternatives are desperately needed.
Virotherapy is a relatively novel treatment approach, which harnesses the natural ability of some viruses to kill the cells in which they proliferate and the ability to spread to neighboring cells, thereby amplifying the therapeutic effect of the initial input dose. Requirements of optimal viral vectors include an efficient capability to find specific target cells and express the viral genome in the target cells. Furthermore, therapeutically optimal vectors have to stay active in the target tissues or cells long enough to exert their therapeutic efficacy while causing minimal effects in normal cells. There has been some progress in developing these beneficial properties of therapeutic viral vectors during the last decades and, for example, retroviral, adenoviral and adeno-associated viral vectors have been widely studied in biomedicine.
Contrary to the viral gene therapy approach, in which foreign genetic material is introduced in cells to correct genetic defects, oncolytic virotherapy takes advantage of the similarities between cellular mechanisms of carcinogenesis and DNA virus replication to direct the cell lysing activity of an oncolytic virus to tumor. In virotherapy the cancer cell transduction and viral replication are carefully controlled by genetic engineering of the viral genome to gain effective and safe tumor eradication. In other words, the use of replicating, oncolytic viruses for cancer treatment necessitates introduction of various genetic modifications to the viral genome, thereby restraining replication exclusively to tumor cells and eventually obtaining selective eradication of the tumor without side effects to healthy tissue.
Upon infection, adenoviruses need to induce a cell cycle S-phase-like state in order to transcribe and replicate the viral genome. E1A is the first viral protein to be expressed in a transduced cell and it can activate transcription of other early viral genes by interactions with cellular check point proteins. Importantly, E1A expression results in the activation of the Eta promoter and the E2 region transcription, leading to the expression of adenoviral replication machinery (Berk 1986, Annu Rev Genet 20: 45-79).
Specific deletions on adenoviral key regulatory genes have been utilized to create dysfunctional proteins or the lack of their expression that leads to dependence on a specific genetic feature present in target cells. Partial deletions of E1A result in restricted replication in normal cells but allow replication in target cells, such as cancer cells. Conditionally replicating viruses featuring a 24 base pair deletion in the CR2 (constant region 2) have been created and shown to be potent and selective in the treatment of glioma and breast cancer xenografts (Fueyo et al. 2000, Oncogene 19:2-12; Heise et al. 2000, Nat Med 6:1134-9). Their cancer specificity results from the inability of dysfunctional E1A to release E2F1 transcription factor, which leads to the requirement of free E2F1. E2F1 is abundant in cancer cells, where the pRb pathway is most often disrupted (Hanahan and Weinberg 2000, Cell 100:57-70).
Most clinical trials have been performed with early generation adenoviruses based on adenovirus 5 (Ad5). The anti-tumor effect of oncolytic adenoviruses depends on their capacity for gene delivery. Unfortunately, most tumors have low expression of the main Ad5 receptor, coxsackie-adenovirus receptor (CAR).
Currently most oncolytic viruses in clinical use are highly attenuated in terms of replication due to several deletions in critical viral genes. These viruses have shown excellent safety record, but the antitumor efficacy has been limited. However, clinical and preclinical results show that treatment with unarmed oncolytic viruses is not immunostimulatory enough to result in sustained anti-tumoral therapeutic immune responses. In this regard, oncolytic viruses have been armed to be more immunostimulatory. Virally infected cells are superior at delivery of nonviral antigen (i.e. tumor antigen) for cross-presentation (Schulz et al. 2005, Nature 433:887-92), and virally induced cell death would be expected to enhance the availability of tumor-associated antigens for uptake by dendritic cells (DCs) (Moehler et al. 2005, Hum Gene Ther 16:996-1005) and subsequently enhance stimulation of cytotoxic T-cells. Furthermore, viral infection may alter the balance of cytokine production from the tumor, and subsequently affect the nature of the immune reaction to the tumor, that is, by counteracting the immunosuppressive nature of the tumor microenvironment (Prestwich et al. 2008, Expert Rev Anticancer Ther 8:1581-8). Most importantly, viruses can be engineered to express highly immunogenic proteins such as granulocyte-macrophage colony-stimulating factor (GM-CSF). When immunogenic proteins are expressed within tumor cells, they are potent stimulators of specific and long-lasting antitumor immunity. Introduction of immunotherapeutic genes into tumor cells and, furthermore, their translation into proteins, leads to the activation of the immune response and to more efficient destruction of tumor cells. The most relevant immune cells in this regard are natural killer cells (NK) and cytotoxic CD8+ T-cells.
Adenoviral Vectors
Adenoviruses are non-enveloped viruses 70-90 nm in diameter with an icosahedral capsid. Their genome is linear, double stranded DNA varying between 25-45 kilobases in size with inverted terminal repeats (ITRs) at both termini and a terminal protein attached to the 5′ ends (Russell 2000, J gen Virol 90:1-20).
The icosahedral capsid is formed by three major proteins, of which the hexon trimers are most abundant (Nemerow et al. 2009, Virology 384:380-8). Each of the twelve vertices of the capsid also contains a pentameric protein, a penton base that is covalently attached to the fiber. The fiber is a trimeric protein that protrudes from the penton base and is a knobbed rod-like structure. Other viral proteins Ma, IVa2, VI, VIII and IX are also associated with the viral capsid. The proteins VII, small peptide mu and a terminal protein (TP) are associated with DNA. Protein V provides a structural link to the capsid via protein VI.
All human adenoviruses have similarities in their fiber architecture. Each has an N-terminal tail, a shaft with repeating sequences, and a C-terminal knob domain with a globular structure. The knob domain is principally responsible for binding the target cellular receptor and its globular structure presents a large surface for lateral and apical binding. The fiber proteins of adenoviruses from different subgroups most distinctively differ in length and ability to bend.
The fiber participates in attachment of the virus to the target cell. First, the knob domain of the fiber protein binds to the receptor of the target cell, secondly, the virus interacts with an integrin molecule, and thirdly, the virus is endocytosed into the target cell. Next, the viral genome is transported from endosomes into the nucleus and the replication of the viral genome can begin (Russell W. C. 2000, J General Virol 81, 2573-2604).
Adenoviruses are dependent on the cellular machinery to replicate the viral genome. They can infect quiescent cells and induce them into a cell cycle S-phase-like state enabling viral DNA replication. The adenoviral genome can be divided into immediate early (E1A), early (E1B, E2, E3, E4), intermediate (IX, Iva), and late (L1-L5) genes (Russell 2000).
Adenoviral transcription can be described as a two-phase-event, early and late, characterized by the expression of different viral genes and separated by the onset of viral DNA replication (Russell 2000, J gen Virol 90:1-20). The first transcription unit to be expressed is the E1A. The E1A proteins stimulate the transcription of other early genes and modulate the expression of cellular genes involved in the transition into S-phase, making the cell more susceptible to viral DNA replication (Berk 1986, Annu Rev Genet 20: 45-79). The E1B proteins suppress cell death elicited in response to unregulated cell proliferation signals, including those mediated by E1A (Moran 1993, FASEB J 7:880-5). The E2 gene products provide the replication machinery for viral gene products.
E3 gene products are not essential for virus replication in vitro, but are dedicated to the control of various host immune responses. E3-gp19K inhibits the transport of the class 1 major histocompatibility complex (MHC) from the endoplasmic reticulum (ER) to the plasma membrane, thereby preventing the presentation of peptides to T lymphocytes by MHC (Rawle et al. 1989, J Immunol 143:2031-7). Other E3 proteins inhibit apoptosis elicited by various cellular proteins such as the tumor necrosis factor α (TNFα) (Wold 1993, J Cell Biochem 53:329-35). As an exception, E3 derived adenoviral death protein (ADP) functions late in the viral cycle to promote cell death, presumably to aid in the release of the virus after all the replicative functions have been completed. E4 gene products have been implicated in many events that occur as the late program begins. E4 proteins augment viral DNA synthesis and messenger RNA (mRNA) transport, late viral gene expression, shutoff of host protein synthesis, and production of progeny virions. The late gene transcription leads to the production of viral structural components and the encapsidation and maturation of the viral particles in the nucleus.
More than 50 different serotypes of adenoviruses have been found in humans. Serotypes are classified into six subgroups A-F and different serotypes are known to be associated with different conditions i.e. respiratory diseases, conjunctivitis and gastroenteritis. Adenovirus serotype 5 (Ad5) is known to cause respiratory diseases and it is the most common serotype studied in the field of gene therapy. In the first Ad5 vectors E1 and/or E3 regions were deleted enabling insertion of foreign DNA to the vectors (Danthinne and Imperiale 2000, Gene Ther 7:1707-14). Furthermore, deletions of other regions as well as further mutations have provided extra properties to viral vectors. Indeed, various modifications of adenoviruses have been suggested for achieving efficient anti-tumor effects.
EP1377671 B1 (Cell Genesys, Inc.) and application US2003/0104625 A1 (Cheng C. et al.) describe an oncolytic adenoviral vector encoding an immunotherapeutic protein granulocyte-macrophage colony-stimulating factor (GM-CSF).
EP1767642 A1 (Chengdu Kanghong Biotechnologies Co., Ltd.) discloses oncolytic adenoviral vectors having improved effects on human immune response.
WO2010072900 discloses oncolytic adenoviral vectors having a modified viral genome and an immunostimulatory GM-CSF.